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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 02 August 2016 and 05 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
(EC) No 440/2008, of 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
Clear, colorless

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium.
Cell source:
other: Not specified as study used an EpiDerm™ Reconstructed Human Epidermis Model Kit supplied by MatTek
Source strain:
other: Not applicable
Details on animal used as source of test system:
Not applicable
Justification for test system used:
Recognised in vitro test for corrosivity
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 23348
- Delivery date: 02 August 2016
- Date of initiation of testing: 02 August 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37”C
- Temperature of post-treatment incubation: Room temperature.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: . Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm

NUMBER OF REPLICATE TISSUES: 2

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL
- Concentration (if solution): used as supplied

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL Sterile ditilled water
- Concentration (if solution): not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): 8.0N Potassium Hudroxide
Duration of treatment / exposure:
3 and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 Minute exposure
Value:
ca. 127.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No indication of corrosion
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 Minute exposure
Value:
ca. 123.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No indication of corrosion
Other effects / acceptance of results:
Quality Criteria
The mean OD562 for the negative control treated tissues was 1.656 for the 3 Minute exposure period and 1.596 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 5.4% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

The relative mean viabilities for each treatment group were as follows:

 Exposure Period  Percentage Viability       
   Negative Control  Positive Control  Test Item
 3 minute  100*  5.5  127.4
 60 minute 100*   5.4  123.3

*The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be non-corrosive to the skin.
Executive summary:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue.  Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control.  The results are used to make a prediction of the corrosivity potential of the test item.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes.  Negative and positive control groups were treated for each exposure period.  At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT loading.  After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 L samples were transferred to the appropriate wells of a pre-labeled 96 well plate.  The optical density (OD) was measured at 562 nm (OD562).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viabilities for each treatment group were as follows:

 Exposure Period  Percentage Viability       
   Negative Control  Positive Control  Test Item
 3 minute  100*  5.5  127.4
 60 minute 100*   5.4  123.3

*The mean viability of the negative control tissues is set at 100%

Quality criteria:  

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was considered to be non-corrosive to the skin.